Nakagawa Takahito, Hashimoto Reisuke, Maruyama Kohei, Tanaka Tsuyoshi, Takeyama Haruko, Matsunaga Tadashi
Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16, Naka-cho, Koganei, Tokyo 184-8588, Japan.
Biotechnol Bioeng. 2006 Aug 5;94(5):862-8. doi: 10.1002/bit.20904.
Bacterial magnetic particles (BMPs) were modified with 3-[2-(2-aminoethylamino)-ethylamino]-propyltrimethoxysilane (AEEA) to produce a dense amine surface. Modification of BMPs in a toluene solution resulted in an increased amine yield, and approximately 11.3 x 10(4) surface amines were detected on a single particle. The modified BMPs were capable of efficient electrostatic capture of DNA. The maximum amount of DNA captured on 10 microg of aminosilane-modified BMPs was 600 ng. A 10 mM phosphate buffer effectively released the captured DNA. This efficiency was dramatically enhanced by incubation at 80 degrees C and DNA recovery from aminosilane-modified BMPs approached 95%. DNA extraction from whole blood using these modified BMPs, followed by PCR, was successfully performed. Furthermore, automated single nucleotide polymorphism (SNP) detection of the aldehyde dehydrogenase 2 (ALDH2) was demonstrated.
用3-[2-(2-氨基乙氨基)-乙氨基]-丙基三甲氧基硅烷(AEEA)对细菌磁性颗粒(BMPs)进行修饰,以产生致密的胺表面。在甲苯溶液中对BMPs进行修饰可提高胺产量,并且在单个颗粒上检测到约11.3×10⁴个表面胺。修饰后的BMPs能够有效地静电捕获DNA。10μg氨基硅烷修饰的BMPs上捕获的DNA最大量为600 ng。10 mM磷酸盐缓冲液可有效释放捕获的DNA。在80℃孵育可显著提高这种效率,从氨基硅烷修饰的BMPs中回收的DNA接近95%。使用这些修饰的BMPs从全血中提取DNA,随后进行PCR,已成功完成。此外,还展示了醛脱氢酶2(ALDH2)的自动化单核苷酸多态性(SNP)检测。